Human Rhcg Ammonia Conduction Mechanism

Computational Molecular Bioscience, 2020, 10, 81-94 https://www.scirp.org/journal/cmb ISSN Online: 2165-3453 ISSN Print: 2165-3445

Human RhCG Ammonia Conduction Mechanism

Henry J. Hoyhtya, Hanna J. Koster, Maggie M. Christiansen, Ugur Akgun*

Physics Department, Coe College, Cedar Rapids, USA

How to cite this paper: Hoyhtya, H.J., Abstract Koster, H.J., Christiansen, M.M. and Akgun, U. (2020) Human RhCG Ammonia Con- The structural differences between E. coli AmtB and the human RhCG as well duction Mechanism. Computational Mo- as the Rh50 from Nitrosomonas europaea suggest different ammonia con- lecular Bioscience, 10, 81-94. duction mechanisms for the AmtB and the Rh proteins. This study investi- https://doi.org/10.4236/cmb.2020.103006 gates the mechanism differences by using molecular dynamics simulations on Received: July 31, 2020 RhCG and Rh50NE structures. Unlike AmtB the Rh proteins lack the aro- Accepted: September 7, 2020 matic cage at the bottom of the periplasmic vestibule. This report establishes Published: September 10, 2020 + the periplasmic Glu residue as the NH4 binding site for Rh proteins, and

Copyright © 2020 by author(s) and the two Phe residues at the entrance of the pore as the NH3 binding site. The Scientific Research Publishing Inc. + This work is licensed under the Creative NH4 molecule pushed by another ammonium releases one of its protons on Commons Attribution International its way to the phenyl gate. This study also discovers that, unlike AmtB, the Rh License (CC BY 4.0). protein pores allow water molecules, which eventually facilitates the NH3 http://creativecommons.org/licenses/by/4.0/ conduction from periplasm to cytoplasm. Open Access

Keywords Rh Proteins, RhCG, Rh50, Rh50NE, Ammonia Channel

1. Introduction

The facilitated diffusion of ammonia across cellular membranes by the Amt/Mep/Rh family is key for acid-base homeostasis. The ability to transport ammonia by the four structures from the Rh/Mep/Amt superfamily was determined using x-ray crystallography of AmtB from Escherichia coli [1] [2], Amt-1 from Archaeoglobus fulgidus [3], Rh50 from Nitrosomonas europaea (Rh50NE) [4], and RhCG from humans [5]. The five human Rhesus proteins fall into two different groups: ammonia transporting glycoproteins (RhAG, RhBG, and RhCG) and the non-transporting Rh proteins (RhD and RhCE) [5]. RhBG and RhCG are found in a variety of tis- sues including the brain, liver, gut, and kidney. Epithelial expressions of RhBG and RhCG in the kidney are involved with ammonium secretion in the connect- ing tubules and collecting ducts [6]. While RhCG has been reported to be found

DOI: 10.4236/cmb.2020.103006 Sep. 10, 2020 81 Computational Molecular Bioscience

H. J. Hoyhtya et al.

in the basolateral and apical membranes; RhBG has not yet been detected in those areas [7]. The previous computational studies for the AmtB mechanism [8] showed that the ammonium initially comes in contact with the charged residues at the perip- + lasmic loops of Escherichia coli. Then, NH4 follows the negatively charged oxygen atoms of the side chain of the carboxyl groups and arrives at the aromatic matic cage, which is composecage at the bottom of the periplasmic vestibule. This arod of F103, F107 and W148, stabilizes the charged substrate at the entrance of the hydrophobic pore. AmtB from Escherichia coli has two tightly stacked phenylalanine rings at the periplasmic entrance of the pore. The synchronized open- ing and closing of these two phenylalanine rings allows charged ammonium to enter the pore and release its proton to the bulk of water, becoming a neutral

NH3 in the transfer [9]. Many studies have been done to decipher if ammonia is transported into the + cell by RhCG as NH4 or as NH3. Measurement of apical plasma membrane + NH4 and NH3 permeability in the collecting duct of mice with global Rhcg de- + letion found decreased NH3 permeability without any change in NH4 permeability [10]. Other studies reconstituted purified human RhCG into liposomes and + demonstrated increased NH3 permeability but no differences in NH4 permeability [5] [11]. Thus, the bulk of evidence suggests Rhcg/RhCG functions as a fa-

cilitated NH3 transporter [12] [13]. The structural differences between AmtB and Rh proteins suggest a variation on this Am conduction mechanism [5]. First of all, the RhCG loops are bigger and contain more negatively charged residues, which could possibly yield better conduction rate. In addition, the W148 is replaced by L170, and F103 is completely missing as it does not have comparable residue in RhCG. The absence of an aromatic cage at the bottom of the vestibule raises the question of recruitment site. At the bottom of the periplasmic vestibule, A144 of AmtB is replaced by a conserved E166 in RhCG, which looks like a prime candidate for a charged substrate recruitment site. The crystal structures of Rh50 from Nitrosomonas europaea and human RhCG also show that the two PHE rings at the periplasmic vestibule exit are not stacked in Rh proteins (see Figure 1). Additionally, the cytoplasmic exit of the pore is not guarded by a PHE ring as in E. Coli AmtB; Hu- man RhCG and Rh50NE have LEU and CYS residues instead. These structural differences at the cytoplasmic exits may result in differences in water’s ability to access the central pores of AmtB and Rh proteins. While this study focuses mainly on RhCG, we also included results from the simulations for Rh50NE to try and investigate general ammonia conduction mechanisms in Rh proteins.

2. Results and Discussion 2.1. E166 Recruits Charged Ammonia

Human RhCG and Rh50NE periplasmic vestibules do not have an aromatic cage [4] [5] [14] as E. coli AmtB does [2]. The main piece of the AmtB aromatic cage,

DOI: 10.4236/cmb.2020.103006 82 Computational Molecular Bioscience

H. J. Hoyhtya et al.

Figure 1. Overlapped view of significant residues in AmtB (red), human RhCG (yellow), and Rh50NE (blue).

W148, is replaced by a LEU in Rh proteins (L126 for Rh50NE, L170 for human + RhCG). The proposed mechanism used by AmtB relies on recruitment of NH4 by the aromatic cage [9], as the absence of W148 signals that Rh proteins use a different mechanism for ammonia recruitment than similar ammonia transporters. At the bottom of the periplasmic vestibule, A144 of AmtB is replaced by a conserved Glu in RhCG (E166) and Rh50NE (E122), which looks like a prime candidate for a charged substrate recruitment site. + Our equilibrium MD simulations showed that NH4 is readily recruited by + E166 on RhCG. The loops of RhCG were particularly effective for NH4 re- + cruitment. Within 5 ns of the equilibration MD simulations two of the NH4 molecules were recruited by E166 from 10 Å away from the lipid phosphate + group plane. This recruitment site attracted the molecules so strongly that NH4 molecules did not leave this site for the next 40 ns of the MD simulations. In one + of the observed recruitment cases, another NH4 molecule was also observed to arrive at the E166 site, and stayed there for almost 20 ns. Our simulations clearly + suggest that conserved E166, which is replaced by A144 in AmtB, is a NH4 recruitment site for RhCG proteins. + We also verified the local energy minima status of E166 site for NH4 by using umbrella sampling simulations. Umbrella sampling simulations also revealed + an energy minimum for NH4 at the phenyl gate. However, we did not observe + NH4 readily moving into the phenyl gate area at any point in 450 ns of a monomeric structure simulation. The energy barrier of around 4 kcal/mol between the E166 residue and the phenyl gate is significant and is likely the main reason that + NH4 does not spontaneously move to phenyl gate energy minima (Figure 2). + One reasonable claim might be that NH4 is being recruited so strongly at + the E166 site that it would prevent any other NH4 molecules from entering the vestibule. However, during our MD simulations, we observed that is not the + + case. Although NH4 is connected to E166 with hydrogen bonds, another NH4 + molecules come as close as 3.9 Å to the recruited NH4 (Figure 3). This means + that the effective charge of the site is not enough to repel other NH4 molecules away from the recruitment site as one might expect; instead the charge of the GLE and the surrounding residues as well as the water molecules “screen” the + + NH4 charge and allow other NH4 molecules to approach.

DOI: 10.4236/cmb.2020.103006 83 Computational Molecular Bioscience

H. J. Hoyhtya et al.

+ Figure 2. Free energy of systems with NH4 and NH3 as the molecules travel into RhCG protein with reference to E166 and F235, the bottom phenyl of the gate.

+ Figure 3. Two NH4 molecules attracted to GLU-166 with a distance of 3.87 Å between them.

+ Although the equilibrium MD simulations have not observed an NH4 ar-

riving at the phenyl gate, NH3 has been observed to position itself comfortably

there, shown in Figure 4. F130 and F235 create an excellent NH3 recruitment

site for RhCG. At this position there is no water within 5 Å of NH3. The free

energy profile derived from the umbrella sampling simulations with NH3 also

supports this result, as the energy minima of NH3 lies just above the bottom phenyl of the gate (Figure 2). + Figure 5 shows the normalized probability distributions for NH3, NH4 and

CO2 molecules through the channel. This distribution, obtained from high den- sity equilibrium simulations, clearly identifies the energy minima locations of + E166 for NH4 and PHE rings for NH3. During the long equilibration simula-

tions, the CO2 molecules did not show any tendency to diffuse the periplasmic vestibule, same as the AmtB and Rh50NE simulations.

DOI: 10.4236/cmb.2020.103006 84 Computational Molecular Bioscience

H. J. Hoyhtya et al.

Figure 4. NH3 settled in phenyl rings with nearest water molecules.

Figure 5. RhCG probability distributions for three substrates, corrected with available space (radius) measured via hole. The z-position of primary residue(s) each substrate is attracted to have been labeled.

2.2. No Synchronized Phenyl Motion for Rh Proteins

Throughout the known structure of the Amt/Mep/Rh protein family the periplasmic vestibule is separated from the pore with two phenylalanine residues. For the Amt protein, the phenylalanine residues are stacked parallel on top of each other and completely cover the channel. Rh proteins, on the other hand, have phenylalanines that create a right angle with each other with one in a vertical position while the other lies horizontal. In their experimental study, Javelle et.al found out that mutating both phenyls kills the function of AmtB [8]. On the other hand, for human RhCG, single mutation of the phenyl pair substantially

stops the NH3 conduction, but the double mutation keeps the channel almost as active as the WT [15]. The difference between these experimental studies suggests that the role of the phenyl gate is different between Rh proteins and AmtB. The conserved twin phenyl residues that keep the gate of the hydrophobic pore closed are observed to flip open in a synchronized manner for E. Coli AmtB [9]. Additionally, the dihedral angle Ca-Cb-Cg-Cd1 distribution shows two energetically favorable configurations of AmtB phenyls. The π-π interaction between the phenyl side chains allows these two stable cases [9]. During over 1 μs MD simulations performed on both Rh50NE and human RhCG, we did not observe synchronized phenyl motion. This may suggest that

DOI: 10.4236/cmb.2020.103006 85 Computational Molecular Bioscience

H. J. Hoyhtya et al.

the deprotonation process of Rh proteins is achieved through a different mechanism than that of AmtB. + The NH4 recruitment location of the GLU is around 10 Å away from the phenyl gate; in our previous study we found that the phenyl gate actively takes + part in the deprotonation of NH4 . This is another indication that the Rh pro- + teins have different conduction mechanisms for both NH4 and NH3 than AmtB. Our analysis on dihedral angles Ca-Cb-Cg-Cd1 shows that when there is no substrate in the vicinity of the phenyl gate, the F130 takes dihedral angles of 16 ± 23 or −170 ± 14 while F235 takes 87 ± 14 or −93 ± 12. These numbers represent the mean and the sigma of the Gaussian fit. Having exactly 180 degrees between these angles clearly shows that there is an only one energy minimum for phenyl pairs on human RhCG in the absence of substrate. However, the distribution of + the same dihedral angles with the presence of NH4 at the E166 recruitment + site shows that the presence of NH4 changes the dihedral angles F130 favors. + When NH4 is in the vicinity, F130 takes two dominant angles: −42 ± 14 and 76 ± 14 (Figure 6). The existence of charged substrates seems to change F130 dynamics while F235 is not affected. Although the crystal structures of Rh50NE and human RhCG manifest similar positions for phenyl pairs, the dynamics of the ring create differences in position frequency. When there is no substrate in the vicinity of the phenyl pairs, the F86 of Rh50NE takes dihedral angles of −61 ± 16 and 60 ± 28 while F194 distribution yields −133 ± 26 and 66 ± 26. Having 180 degrees between the dihedral angles of F194 means the bottom of the phenyl pairs on both human RhCG and Rh50NE have only one energetically favorable position. On the other hand, the top phenyl of Rh50NE (F86) clearly shows two distinct favorable angles with almost 90 degrees between them.

Figure 6. RhCG dihedral angles for F130 (top) and F235 (bottom) (left column), RhCG + F130 (top) and F235 (Bottom) dihedral angles with NH4 is recruited by the protein.

DOI: 10.4236/cmb.2020.103006 86 Computational Molecular Bioscience

H. J. Hoyhtya et al.

+ When we extend the analysis to the cases when the substrate ( NH4 or NH3) is close to the phenyls, the top phenyl shows different angle preference. The F86 residue prefers to keep the Ca-Cb-Cg-Cd1 dihedral angle at −61 ± 16 minima, while F194 seems unaffected by the existence of the substrate.

2.3. Water Leaks into the Central Pore of Rh Proteins

The crystal structures of Rh50NE [4] [14] (1.3 Å resolution) and human RhCG (2.1 Å resolution) have some distinct pore differences when compared to E. coli AmtB. The conserved phenyl rings at the pore entrance from the periplasmic vestibule are not stacked as tightly as in AmtB. The cytoplasmic exits of the pores on these structures do not have a phenyl gate either. This allows water molecules to have hydrogen bonds with central histidine residues, thus shorten- ing the length of the hydrophobic pore to 11 Å. By contrast, the distance between the water molecules on both sides of the pore for AmtB is more than 20 Å. During long equilibrium MD simulations (equivalent of 540 ns simulations for a monomer) on a trimeric Rh50NE model, water molecules were observed to diffuse through pore in both directions. Although the F194 appears to keep the periplasmic entrance of Rh50NE pore closed, there is still enough space for water molecules to diffuse through. Throughout the simulations, a total of 16 water molecules leaked into the pore. Four of these water molecules diffused from the cytoplasmic vestibule to the periplasmic vestibule, and four others diffused in the opposite direction. The remaining 8 water molecules were observed to leak into pore from cytoplasm; after spending some time in pore they moved back to the cytoplasmic vestibule. The average time it took for a water molecule to diffuse from periplasm to cytoplasm was measured to be 2.4 ns. The cytoplasmic entrance of the pore is even less protected for Rh50NE proteins. The four water molecules conducted to the periplasmic vestibule of Rh50NE from cytoplasmic vestibule in average of 1.1 ns. However, the rest were seen going back to the cytoplasmic vestibule after spending an average of 1.5 ns within the pore. On two different occasions two water molecules entered together to Rh50NE pore. The crystal structure of Rh50NE shows that the bottom of the periplasmic vestibule is divided into two parts by the I198 and F86. The narrow pathway created by A195, T196, A197, and I198 allows water molecules to create a water bridge between the pore entrance and the periplasmic vestibule. The water conduction from periplasm to pore is mediated through this narrow pore. Other- wise the water molecules located at the entrance of the pore have not been observed to be accessible from the F86 side of the periplasmic vestibule. Chérif-Zahar

et al. [16] found that Rh50NE regulates NH3 uptake, but that it appears to lack the primary proposed ammonium binding site found in AmtB and thus seems to function better in a higher pH environment. A computational study by Hub et al. [17] also reports that the Rh50NE should be partially active and suggests that

its water conduction rate is 40 times slower than its conduction rate of NH3. The 540 ns monomeric MD simulations reported here clearly observed that water molecules being conducted by Rh50NE.

DOI: 10.4236/cmb.2020.103006 87 Computational Molecular Bioscience

H. J. Hoyhtya et al.

In regards to the human RhCG model, Baday et al. [18] believed the pore to be hydrophobic because of a conserved phenylalanine. Our simulations show that, although the water molecules can enter the channel, the human RhCG does not conduct water molecules. After approximately 450 ns of monomeric MD simulations performed with a trimeric human RhCG model, we observed 23 water molecules entering the pore from the cytoplasmic vestibule. All of these water molecules leaked into the pore from the cytoplasmic vestibule and returned back to cytoplasm. During the water leakages to the pore, the water molecule(s) tended to stay in the pore for 5 ns or less (Figure 7). As is shown by the crystal structure, there is a water molecule at the top of the pore that has hydrogen bonds with H185, G179, G290 and N236 and does not leave. Unlike Rh50NE, this water molecule positioned at the pore and periplasm interface does not have any direct connection to the bulk of the water in the periplasmic vestibule. How- ever, the cytoplasmic entrance of the human RhCG pore allows water molecules to leak through more freely. On many occasions during 450 ns monomeric MD simulations, two water molecules were observed to enter the pore together. In these cases, the water molecules were more stable inside the pore. Interestingly, in one occasion we observed a transiently stable chain of water molecules flood- ing the pore from cytoplasm all the way to F235 (Figure 8).

3. Conclusions and Discussion

In an attempt to better understand the mechanism of transport in RhCG transporters, we performed various simulations that affected different molecules in the system to better identify how they interact. Although the transport mechanism is still a mystery, our simulations suggest + some viable theories as to what the process is. Because NH4 is attracted to

E166 and NH3 is attracted to the phenylalanines, it is logical to assume that there + is a place of deprotonation somewhere between the two points. Another NH4

Figure 7. Positions of the water molecules interacting with the RhCG pores over time. The two lines represent F235 (left) and H334 (right).

DOI: 10.4236/cmb.2020.103006 88 Computational Molecular Bioscience

H. J. Hoyhtya et al.

Figure 8. Water chain observed in the periplasmic pore of RHCG. The water molecules enter from cytoplasm but they cannot pass to periplasm. W490 was detected at this location on the x-ray structure of the protein [5].

+ molecule has been observed to come very close to the NH4 molecule already attracted to the E166. Since there are no proton acceptors around it, this suggests the deprotonation happens before the rings, possibly between E166 and the phenyl gate. The new molecule may serve as a catalyst to kick out the already at- + tracted NH4 molecule, causing it to be forced over the energy barrier. Because of the large barrier of around 4 kcal/mol beyond E166, it is reasonable + to believe that the NH4 molecule loses a proton before making the jump, con- sidering it would be more energetically favorable to break through the barrier in an uncharged state. It would be very difficult for it to get over the barrier with-

out doing so. After it loses the proton and becomes NH3, it is attracted to the phenylalanines at the top of the pore. The phenylalanines are constantly moving and turning in the simulations.

This might give the NH3 molecule the opportunity to slip into the channel and navigate to the pocket with the structural water molecule. Once in the channel,

we know that the NH3 will act as the water does because it is a similar size, and will jump down past the histidines into the periplasm (Figure 9). During our

simulations we substituted a NH3 molecule entering the pore in place of the

second water molecule, and observed that the NH3 acted similarly to water and had jumped down the pore to the cytoplasmic side. Our simulations have shown that the pore of RhCG is much less hydrophobic than that of AmtB. During our long equilibrium MD simulations (the equivalent of 740 ns for a single monomer) in an earlier study on trimeric AmtB, the author of this study reported only one water molecule entering the hydrophobic pore from cytoplasmic vestibule [9], unlike another study reporting hydrophilic AmtB pore [19]. In this study, we found that both the Rh50NE and human RhCG crystal structures had water molecules at the cytoplasmic exits of the pore. Further- more, Zidi-Yahiaoui et al. report water conduction by human RhCG in HEK293E cells [14]. The reported conduction rate is ten times less than AQP1 and does not change with F130A/F235V double mutation. However, Gruswitz et al. reports that stopped flow assays conducted on proteoliposomes do not show any water conduction by human RhCG. During our 450 ns of monomeric MD si-

DOI: 10.4236/cmb.2020.103006 89 Computational Molecular Bioscience

H. J. Hoyhtya et al.

mulations we did not see any water conduction from one vestibule to the other. These results are in agreement with Gruswitz et al. [5]. Further simulations and investigation will be necessary to illustrate the com- plete transport mechanism in RhCG. Nonetheless, our simulations suggest that + NH4 is brought in and is converted into NH3, which then slips into the pore and diffuses in the same way the water molecules do. Figure 10 summarizes our proposed mechanism of human RhCG ammonia conduction.

Figure 9. Our simulations showed NH3 acting like a water molecule when we placed it in the channel. On the left, it is stuck in the pore with the first water molecule and on the right it is shown to move down the histidine complex as expected.

+ Figure 10. Proposed mechanism of NH4 deprotonation and NH3 conduction by RhCG. + + (a) NH4 is recruited by E166; (b) A second NH4 molecule is attracted to E166, push- + + ing the first NH4 towards the phenyl rings; (c) The first NH4 reaches the energy bar-

rier, sheds a proton to become NH3 and settles into the phenyl rings at its energy mini-

mum; (d) As the rings fluctuate, the NH3 works its way into the pore, and settles near the

water shown in the crystal structure in the mildly unstable pocket; (e) NH3 acts in a simi-

lar manner to water, and rushes out of the pore down the channel; (f) NH3 is flushed out of the channel.

DOI: 10.4236/cmb.2020.103006 90 Computational Molecular Bioscience

H. J. Hoyhtya et al.

4. Materials and Methods

Model building. The trimeric human RhCG model was based upon the X-ray structure of RhCG at 2.1 Å resolution (PDB ID = 3HD6) [5]. The periplasmic loops with 18 residues (residue IDs 35 - 52, and 22 residues (residue IDs 362 - 283) were constructed by using COOT [20] [21]. The trimeric Rh50 from Ni- trosomonas europaea model was based upon the X-ray structure with 1.3 Å resolution (PDB ID = 3B9W) [14]. The trimeric AmtB model was based upon the X-ray structure of AmtB at 1.35 Å resolution (PDB ID = 1U7G) [2]. Three mu- tations (F68S, S126P, and K255L) and SeMet residues were changed back to their native residues. Hydrogen atoms were added to the structures with XPLOR-NIH [22]. The protonation states for each His residue were modeled based on their hydrogen bond environment. Phosphatidylethanolamine (POPE) was used to create a model of a lipid bilayer with dimensions of 120 Å × 120 Å × 39 Å. Trimeric protein was inserted into the center of the lipid bilayer and overlapping POPE molecules were removed. The z-axis is aligned in the direction of the hydrophobic pore of the protein and normal to the lipid bilayer plane. The lipid-protein complex was solvated by adding 25 Å TIP3P [23] water slabs on each side of the lipid bilayer. The model used in all simulations contains 22,718 water molecules and more than 142,000 atoms, with dimensions of 120 Å × 120 Å × 86 Å. All the MD simulations were performed with the NAMD program [24] using the CHARMM36 force field [25] [26]. Periodic boundary conditions were applied in all three dimensions. A Langevin thermostat and the Langevin piston model [27] were used to keep the temperature and pressure constant at 310 K and 1 atm, respectively. The Van der Waals interaction cutoff was set at 12 Å in conjunction with switching at 8 Å. Full electrostatics was employed using the Particle Mesh Ewald (PME) method [28], with a grid spacing less than 1 Å. All simulations used an integration time step of 1.0 fs. The total charge of the system was neutralized by adding Cl− and Na+ ions. Equilibrium molecular dynamic simulations. To investigate the water diffusion into protein vestibules, separate initial models were built for each protein. Before the long MD simulations, each system was first minimized for 2000 steps and then equilibrated for 2 ns with no constraints on the system. Total of 246 ns, 166 ns, and 164 ns of MD simulations were performed on AmtB, Rh50NE, and RhCG, respectively. To determine how water diffuses into these proteins, the probability distributions were calculated, by tracing the 22,718 water molecules inside a hypothetical cylinder, perpendicular to the plane of the lipid bilayer. The height of the cylinder extends the thickness of the periodic boundary box, and with 70 Å di- ameter it accommodates the trimeric protein. The number of solute molecules that appeared inside the cylindrical volume was recorded at every 5 ps frames by monitoring the location of the oxygen atom of each water during the MD simulations. The probability distribution curves were derived from the observed num-

DOI: 10.4236/cmb.2020.103006 91 Computational Molecular Bioscience

H. J. Hoyhtya et al.

ber of solute molecules per ns × Å3 along the axis of the cylinder. The probability distributions were corrected with respect to the radius of the channel at that z value. The radius of the channels was determined by using HOLE program. For each protein, the total probability distributions within the cylinder were normalized to 100. Umbrella sampling simulations. The umbrella sampling methods were used to

calculate the free energy profile of H2O conduction through proteins. During umbrella sampling simulations, each monomer was sampled simultaneously with a 0.5 Å window size. In each sampling window atom coordinates were recorded at every 0.5 ps, for 450 ps. The first 150 ps were used for equilibration and the last 300 ps were defined as the production run. The starting point for each window was determined by the positions of the atoms from the last frame of the previous window. This way sufficient relaxation of the system was assured. Å bi- asing harmonic potential with force constants of 10 kcal/(mol × Å2) was applied

to the O of the H2O molecule. The solute molecules were restrained only on the z axis and free to move on the xy plane. The data were unbiased and a free energy profile was calculated by using Weighted Histogram Analysis (WHAM) method [29] [30].

Acknowledgements

The authors would like to thank XSEDE for computational time, Coe College, and the McElroy Foundation for continuous support. This work has been sup- ported by NSF grants PHY1950337 and DMR1746230.

Conflicts of Interest

The authors declare no conflicts of interest regarding the publication of this paper.

References [1] Zheng, L., Kostrewa, D., Berneche, S., Winkler, F.K. and Li, X.D. (2004) The Me- chanism of Ammonia Transport Based on the Crystal Structure of AmtB of Esche- richia coli. Proceedings of the National Academy of Sciences of the United States of America, 101, 17090-17095. https://doi.org/10.1073/pnas.0406475101 [2] Khademi, S., et al. (2004) Mechanism of Ammonia Transport by Amt/MEP/Rh: Structure of AmtB at 1.35 A. Science, 305, 1587-1594. https://doi.org/10.1126/science.1101952 [3] Andrade, S.L.A., Dickmanns, A., Ficner, R. and Einsle, O. (2005) Crystal Structure of the Archaeal Ammonium Transporter Amt-1 from Archaeoglobus fulgidus. Pro- ceedings of the National Academy of Sciences of the United States of America, 102, 14994-14999. https://doi.org/10.1073/pnas.0506254102 [4] Li, X., Jayachandran, S., Nguyen, H.H. and Chan, M.K. (2007) Structure of the Ni- trosomonas europaea Rh Protein. Proceedings of the National Academy of Sciences of the United States of America, 104, 19279-19284. https://doi.org/10.1073/pnas.0709710104

DOI: 10.4236/cmb.2020.103006 92 Computational Molecular Bioscience

H. J. Hoyhtya et al.

[5] Gruswitz, F., et al. (2010) Function of Human Rh Based on Structure of RhCG at 2.1 A. Proceedings of the National Academy of Sciences of the United States of Ameri- ca, 107, 9638-9643. https://doi.org/10.1073/pnas.1003587107 [6] Zidi-Yahiaoui, N., Mouro-Chanteloup, I., D’Ambrioso, A., et al. (2005) Human Rhesus B and Rhesus C Glycoproteins: Properties of Facilitated Ammonium Trans- port in Recombinant Kidney Cells. Biochemical Journal, 391, 33-40. https://doi.org/10.1042/BJ20050657 [7] Wagner, C., Devuyst, O., Belge, H., Bourgeois, S. and Houillier, P. (2010) The Rhe- sus Protein RhCG: A New Perspective in Ammonium Transport and Distal Urinary Acidification. Kidney International, 79, 154-161. https://doi.org/10.1038/ki.2010.386 [8] Javelle, A., Lupo, D., Ripoche, P., Fulford, T., Merrick, M. and Winkler, F.K. (2008) Substrate Binding, Deprotonation, and Selectivity at the Periplasmic Entrance of the Escherichia coli Ammonia Channel AmtB. Proceedings of the National Academy of Sciences of the United States of America, 105, 5040-5045. https://doi.org/10.1073/pnas.0711742105 [9] Akgun, U. and Khademi, S. (2011) Periplasmic Vestibule Plays an Important Role for Solute Recruitment, Selectivity, and Gating in the Rh/Amt/MEP Superfamily. Proceedings of the National Academy of Sciences of the United States of America, 108, 3970-3975. https://doi.org/10.1073/pnas.1007240108 [10] Biver, S., Belge, H., Bourgeois, S., et al. (2008) A Role for Rhesus Factor Rhcg in Renal Ammonium Excretion and Male Fertility. Nature, 456, 339-343. https://doi.org/10.1038/nature07518 [11] Attmane-Elakeb, A., Amlal, H. and Bichara, M. (2001) Ammonium Carriers in Medullary Thick Ascending Limb. American Journal of Physiology-Renal Physiol- ogy, 280, F1-F9. https://doi.org/10.1152/ajprenal.2001.280.1.F1

+ [12] Weiner, D. and Verlander, J. (2010) Role of NH3 and NH4 Transporters in Renal Acid-Base Transport. American Journal of Physiology-Renal Physiology, 300, F11-F23. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3023229/#B38 [13] Weiner, D. and Verlander, J.W. (2010) Molecular Physiology of the Rh Ammonia Transport Proteins. Current Opinion in Nephrology and Hypertension, 19, 471-477. https://doi.org/10.1097/MNH.0b013e32833bfa4e [14] Lupo, D., Li, X., Durand, A., et al. (2007) The 1.3-A Resolution Structure of Nitro-

somonas europaea Rh50 and Mechanistic Implications for NH3 Transport by Rhe- sus Family Proteins. Proceedings of the National Academy of Sciences of the United States of America, 104, 19303-19308. https://doi.org/10.1073/pnas.0706563104 [15] Zidi-Yahiaoui, N., Callebaut, I., Genetet, S., et al. (2009) Functional Analysis of Human RhCG: Comparison with E. coli Ammonium Transporter Reveals Similari- ties in the Pore and Differences in the Vestibule. American Journal of Physiology: Cell Physiology, 297, C537-C547. https://doi.org/10.1152/ajpcell.00137.2009 [16] Chérif-Zahar, B., Durand, A., Schmidt, I., Hamdaoui, N., Matic, I., Merrick, M. and Matassi, G. (2007) Evolution and Functional Characterization of the RH50 Gene from the Ammonia-Oxidizing Bacterium Nitrosomonas europaea. Journal of Bacte- riology, 189, 9090-9110. https://doi.org/10.1128/JB.01089-07 [17] Hub, J.S., Winkler, F.K., Merrick, M. and de Groot, B.L. (2010) Potentials of Mean Force and Permeabilities for Carbon Dioxide, Ammonia, and Water Flux across a Rhesus Protein Channel and Lipid Membranes. Journal of the American Chemical Society, 132, 13251-13263. https://doi.org/10.1021/ja102133x

DOI: 10.4236/cmb.2020.103006 93 Computational Molecular Bioscience

H. J. Hoyhtya et al.

[18] Baday, S., Shihao, W., Lamoureux, G. and Bernèche, S. (2013). Different Hydration Patterns in the Pores of AmtB and RhCG Could Determine Their Transport Me- chanisms. Biochemistry, 52, 7091-7098. https://doi.org/10.1021/bi400015f [19] Lamoureux, G., Klein, M.L. and Berneche, S. (2007) A Stable Water Chain in the Hydrophobic Pore of the AmtB Ammonium Transporter. Biophysical Journal, 92, L82-L84. https://doi.org/10.1529/biophysj.106.102756 [20] Emsley, P., Lohkamp, B., Scott, W.G. and Cowtan, K. (2010) Features and Devel- opment of Coot. Acta Crystallographica Section D Biological Crystallography, 66, 486-501. https://doi.org/10.1107/S0907444910007493 [21] Emsley, P. and Cowtan, K. (2004) Coot: Model-Building Tools for Molecular Graphics. Acta Crystallographica Section D Biological Crystallography, 60, 2126-2132. https://doi.org/10.1107/S0907444904019158 [22] Schwieters, C.D., Kuszewski, J.J., Tjandra, N. and Clore, G.M. (2003) The Xplor-NIH NMR Molecular Structure Determination Package. Journal of Magnetic Resonance, 160, 65-73. https://doi.org/10.1016/S1090-7807(02)00014-9 [23] Jorgensen, W.L., Chandrasekhar, J., Madura, J.D., Impey, R.D. and Klein, M.L. (1983) Comparison of Simple Potential Functions for Simulating Liquid Water. The Journal of Chemical Physics, 79, 926-935. https://doi.org/10.1063/1.445869 [24] Phillips, J.C., Braun, R., Wang, W., et al. (2005) Scalable Molecular Dynamics with NAMD. Journal of Computational Chemistry, 26, 1781-1802. https://doi.org/10.1002/jcc.20289 [25] MacKerell, A.D., Bashford, D., Bellott, M., et al. (1998) All-Atom Empirical Poten- tial for Molecular Modeling and Dynamics Studies of Proteins. The Journal of Physical Chemistry B, 102, 3586-3616. https://doi.org/10.1021/jp973084f [26] Schlenkrich, M., Brickmann, J., MacKerell, A.D. and Karplus, M. (1996) An Empir- ical Potential Energy Function for Phospholipids: Criteria for Parameter Optimiza- tion and Applications. In: Merz, K.M. and Roux, B., Eds., Biological Membranes, Birkhäuser, Boston, 31-81. https://doi.org/10.1007/978-1-4684-8580-6_2 [27] Feller, S.E., Zhang, Y.H., Pastor, R.W. and Brooks, B.R. (1995) Constant Pressure Molecular Dynamics Simulation the Langevin Piston Method. The Journal of Chemical Physics, 103, 4613-4621. https://doi.org/10.1063/1.470648 [28] Essmann, U., Perera, L., Berkowitz, M.L., Darden, T., Lee, H. and Pedersen, L.G. (1995) A Smooth Particle Mesh Ewald Method. The Journal of Chemical Physics, 103, 8577. https://doi.org/10.1063/1.470117 [29] Kumar, S., Rosenberg, J., Bouzida, D., Swendsen, R. and Kollman, P. (1995) Multi- dimensional Free-Energy Calculations Using the Weighted Histogram Analysis Method. Journal of Computational Chemistry, 16, 1339-1350. https://doi.org/10.1002/jcc.540161104 [30] Roux, B. (1995) The Calculation of the Potential of Mean Force Using Computer Simulations. Computer Physics Communications, 91, 275-282. https://doi.org/10.1016/0010-4655(95)00053-I

DOI: 10.4236/cmb.2020.103006 94 Computational Molecular Bioscience